Exploring the basis of heterogeneity of cancer aggressiveness among the mutated POLE variants

Mechanisms and consequences of POLE mutations in human cancers.


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2. The end of this section (page6) contains this sentence, "Several POLE variants within the exonuclease domain such P286R, V411L, L424V, S459F, P286H, F367S and L424I show a decrease in exonuclease activity, as measured by biochemistry experiments using purified Polε."There are no references for this statement (this is a review article, accurate references are essential) and it seems out of context, the authors should describe more about these variants in this section, or leave it to a later section but this one sentence alone seems incomplete, and needs more discussion.In addition, the section above this sentence is all about germline variants, however the variants listed in this sentence are found in somatic POLE mutations, this should be made clear.Heterogeneity of the POLE mutation impact 3. Major Criticism: Again, not including references or citing the earlier work is a major weakness for this review, for example on page 7, the authors do not include all the references they can for many statements, "Besides the DNA mismatch repair defects that underlie Lynch syndrome, the mutations in POLE highlight the critical role of replication errors in predisposition to colorectal and endometrial cancers.This is in contrast to cancers of the breast and ovary, in which double-stranded DNA break repair is more significant in predisposition."There are many references to support this statement, however none are present.Review articles are a good source of finding important references, however this one does not include a large amount and misses that opportunity in multiple sections.4. The title for this section implies they will discuss more details about the different variants, however this section does not do that.This section would benefit from a larger discussion about what is currently known about the variants.This entire section can be expanded to go into detail about each variant.
Mutator phenotype and mutation signature in POLE variants 5. Major Criticism: Again, this section falls short of including references, for example on page 8, this sentence is lacking appropriate references only including later citations while ignoring earlier references (17,59,60) "a high increase in mutation rates have been documented in cancers with P286R, D275V, P286H, F367S, L424V, P436R, and S459F changes located closed to the DNA binding cleft of Pol (31,32)".And again, this sentence "Importantly, all of these variants lead to a much higher mutation frequency as compared to the variant that completely eliminates proofreading" is included without any references .Again, "Among hypermutated cancers presenting {greater than or equal to} 10 Mut/Mb, approximately 25% are associated with mutations in MMR genes alone, whereas a large number of ultra-hypermutated cancers ({greater than or equal to} 100 Mut/Mb) show mutations affecting both MMR and the replicative DNA polymerases, mainly POLE ", is missing citations.Again, on page 8, "Specific signature errors have been shown to be enriched in mutated POLE cancers.These include three trinucleotide hotspot mutations: C>A transversions in TCT context (C>A-TCT), C>T-TCG and T>G-TTT " should include references 17, 15, 59. 6.They should state what types of cancers have MMR and POLE mutations together, which types of cancer that does not occur, and in general expand this section to have a comprehensive review.
Mutated POLE tumors and immunogenic response 7. Major Criticism: This sentence mixes up very different types of high mutation load tumors, and consistent with other sections of this manuscript is missing citations."It is becoming increasingly clear that cancers with high intrinsic mutation load (MMR and POLE mutated cancers) or cancers related to mutagenic environmental genotoxic exposure such lung, melanoma and bladder cancers, which show all a high mutational burdens, respond generally well to immunotherapy." This entire section makes very bold statements with little to no references to back them up.
In this section, as in the rest of the manuscript, they do not make a clear distinction between somatic variants and germline.They discuss their N363K variant with no distinction that they are now describing a germline variant.There are recent papers examining POLE variants and response to therapeutics, in particular, (PMID: 34250404) is not included in this section, yet is a recent description of POLE variants and immune response.
In general, this review could benefit from a more complete survey of the current and past literature, adding relevant references, and expanding some sections as noted above.
Reviewer #2 (Comments to the Authors (Required)): This is a well-written, concise review of the mechanisms and consequences of the different POLE mutations in cancer.The authors also propose some mechanistic basis underlining the mutation heterogeneity and discuss novel considerations for the choice and efficacy of therapies for POLE mutated tumors.My only comment is not a mandatory request.I leave it to the authors to decide if they would like to add a short mention in the section "POLE mutation and cancer" of the recent study on single-strand events that could indicate how POLE mutations arise in cancer.They could refer to the publication of Gilad Evrony (Mei Hong Liu et al. 2023 bioRxiv doi: https://doi.org/10.1101/2023.02.19.526140. Reviewer #3 (Comments to the Authors (Required)): In review, the manuscript presents a summary of current literature on mammalian DNA polymerase involved in the replication of their leading and lagging strand.The authors have assembled and analyzed the current mutations in DNA polymerase epsilon and delta.In part they consider the association between mutant DNA polymerases and cancer.
The major conclusion of the paper is that mutations in these DNA polymerases are associated with specific types of human cancers.The data is well supported but does not necessarily indicate that the association is causative.The paper indicates that human tumors contain large numbers of different mutations.While it would be easy to suggest additional experiments, these would involve many laboratories and many years of research.
I suggest that the authors consider adding two columns to table one stating the locations of each of the mutations, and the second column indicating the enhancement of the mutation frequencies that have been reported.In addition, a clearer distinction between initiation and promotion of carcinogenesis appearing earlier in the paper may be useful.The legend to figure one contains a new original concept.The authors hypothesize that mutations in DNA polymerase could result in an enhanced mutagenesis without altering base specificity.In particular, studies on mutant polymerases may provide a new target for treatment options for specific cancers.This section starts with a description of MMR deficient cancers, then progresses into a description of POLE deficient cancers.The references for many sentences are incomplete or missing, here are some examples, (page 5) "POLE mutations have also been detected, albeit less frequently, in other types of gastrointestinal cancer, as well as in brain, breast, ovary, prostate, lung, kidney, cervix, and bone tumors (13,(20)(21)(22)."does not include earlier papers that initially describe the variants, such as (17,59,60) and does reference databases such as COSMIC, cbioportal which is a bit confusing.Not including the appropriate references is a major problem throughout this manuscript.
We have now included in the entire paragraph appropriate references as required by the reviewer, especially for the types of cancers for which POLE has been found mutated (Campbell et al., 2017;Cancer Genome Atlas, 2012;Cancer Genome Atlas Research et al, 2013;Cerami et al, 2012;Forbes et al, 2015;Grossman et al, 2016;Shinbrot et al., 2014) (see page 5).
2. The end of this section (page6) contains this sentence, "Several POLE variants within the exonuclease domain such P286R, V411L, L424V, S459F, P286H, F367S and L424I show a decrease in exonuclease activity, as measured by biochemistry experiments using purified Polε."There are no references for this statement (this is a review article, accurate references are essential) and it seems out of context, the authors should describe more about these variants in this section, or leave it to a later section but this one sentence alone seems incomplete, and needs more discussion.In addition, the section above this sentence is all about germline variants, however the variants listed in this sentence are found in somatic POLE mutations, this should be made clear.
We apologize for the lack of references regarding the decreased in exonuclease activity of the POLE variants and the lack of clarity on germline vs somatic variants.We have now introduced original references showing that these POLE mutations affect the activity of the exonuclease (Korona et al, 2011;Parkash et al, 2019;Shinbrot et al., 2014) (see page 6).We have also better described the germline versus somatic POLE mutations : see page 5 the paragraph " The last decade has witnessed the identification in cancers from many tissue types of multiple somatically acquired missense mutations clustering in the sequence encoding the exonuclease proofreading domain of POLE……." and page 5-6 the paragraph "Mutations in the exonuclease domain of POLE can also be inherited through the germline, leading to a rare autosomal dominant familial cancer predisposition syndrome documented as polymerase proofreadingassociated polyposis (PPAP), characterized……." Paragraph Heterogeneity of the POLE mutation impact 3. Major Criticism: Again, not including references or citing the earlier work is a major weakness for this review, for example on page 7, the authors do not include all the references they can for many statements, "Besides the DNA mismatch repair defects that underlie Lynch syndrome, the mutations in POLE highlight the critical role of replication errors in predisposition to colorectal and endometrial cancers.This is in contrast to cancers of the breast and ovary, in which double-stranded DNA break repair is more significant in predisposition."There are many references to support this statement, however none are present.Review articles are a good source of finding important references, however this one does not include a large amount and misses that opportunity in multiple sections.
We have also added in this paragraph some aspects on the comparison between POLE and POLD1 mutations in hypermutated cancers in order to make the review more robust (see page 7 the paragraph : "Generally, there are much less cancer driver mutations in POLD1 than in POLE in human cancers.This might be due to the reduced fitness and viability of POLD1 mutants as Pol holds multiple critical roles besides lagging strand replication, including its ability to proofread in trans the errors made by Pol and Pol, its role during MMR and during Okazaki fragment maturation").
4. The title for this section implies they will discuss more details about the different variants, however this section does not do that.This section would benefit from a larger discussion about what is currently known about the variants.This entire section can be expanded to go into detail about each variant.The reviewer is correct and we apologize.As required by the reviewer, we have now expanded the paragraph page 8 by describing more precisely the differential mutagenic impact of the POLE variants in haploid and diploid yeast, and the lack of correlation between the increase of mutation rate, the TMB and the frequency of the variant in tumors.

Major Criticism:
Again, this section falls short of including references, for example on page 8, this sentence is lacking appropriate references only including later citations while ignoring earlier references (17, 59, 60) "a high increase in mutation rates have been documented in cancers with P286R, D275V, P286H, F367S, L424V, P436R, and S459F changes located closed to the DNA binding cleft of Pol (31,32)" As required, we have now included earlier references to the section and again, this sentence "Importantly, all of these variants lead to a much higher mutation frequency as compared to the variant that completely eliminates proofreading" is included without any references .
We apologize, we have now incorporated the paper by Kane and Sherbakova in 2014, which described that cancer-associated POE variants can produce unusually strong mutator phenotype exceeding that of proofreading-deficient mutants by up to two orders of magnitude.
Again, "Among hypermutated cancers presenting {greater than or equal to} 10 Mut/Mb, approximately 25% are associated with mutations in MMR genes alone, whereas a large number of ultra-hypermutated cancers ({greater than or equal to} 100 Mut/Mb) show mutations affecting both MMR and the replicative DNA polymerases, mainly POLE ", is missing citations.
We have now incorporated the appropriate reference by Jansen et al. in 2016.
Again, on page 8, "Specific signature errors have been shown to be enriched in mutated POLE cancers.These include three trinucleotide hotspot mutations: C>A transversions in TCT context (C>A-TCT), C>T-TCG and T>G-TTT "should include references 17, 15, 59.
We have now incorporated these references 6.They should state what types of cancers have MMR and POLE mutations together, which types of cancer that does not occur, and in general expand this section to have a comprehensive review.

Paragraph Mutated POLE tumors and immunogenic response 7. Major Criticism:
This sentence mixes up very different types of high mutation load tumors, and consistent with other sections of this manuscript is missing citations."It is becoming increasingly clear that cancers with high intrinsic mutation load (MMR and POLE mutated cancers) or cancers related to mutagenic environmental genotoxic exposure such lung, melanoma and bladder cancers, which show all a high mutational burdens, respond generally well to immunotherapy." This entire section makes very bold statements with little to no references to back them up.Again, we apologize and we have now incorporated the appropriate reference (Le DT 2015, Le DT 2017, (McGranahan et al, 2016;Rizvi et al, 2015;Yarchoan et al, 2017) In this section, as in the rest of the manuscript, they do not make a clear distinction between somatic variants and germline.They discuss their N363K variant with no distinction that they are now describing a germline variant.We have already modified page 5 for this issue and better described the germline versus somatic POLE mutations.The effect of POLE variant in term of chromosome instability and DNA damage and the associated mechanisms is effective when it is expressed, as somatic or germline, so this is the reason we discussed the general mechanistic aspects independently of the type of the variant.
There are recent papers examining POLE variants and response to therapeutics, in particular, (PMID: 34250404) is not included in this section, yet is a recent description of POLE variants and immune response.We thank the reviewer for this reference that we have now added page 11 (Keshinro et al., JCO Precis Oncol 2021)

Reviewer #2:
We thank this reviewer for his/her global positive appreciation of our review.
We thank also the reviewer for his/her suggestion to add in the section "POLE mutation and cancer" the recent study on single-strand events that could indicate how POLE mutations arise in cancer and refer to the publication of Gilad Evrony (Mei Hong Liu et al. 2023 bioRxiv).This work, which profiled samples from individuals with cancer-predisposition syndromes and defined single-strand mismatch signatures, shows correspondences between single-strand signatures and known double-strand mutational signatures induced by defective proofreading, so this a possible mechanism of the generation of mutation signature in POLE mutants but we have decided not to include it since this mechanistic aspect is not developped in our review, and would require the description of additional alternative mechanisms .

Reviewer #3:
The reviewer proposes to incorporate to table 1 the locations of each of mutation and the corresponding enhancement of the mutation frequencies that have been reported.We thank the reviewer for this suggestion.We have corrected the table 1 with the required items.
The reviewer proposes also to include a clearer distinction between initiation and promotion of carcinogenesis.
We have better described such distinction in page 5 : "Such a defective proofreading activity producing a mutator phenotype, which have been established in model systems, such as yeast, bacteria, and mice, lead to tumorigenesis.These POLE variants are present in heterozygous tumors with no apparent loss of heterozygosity (LOH) and with high mutation loads, up to 500 mutations per megabase (Mut/Mb).A strong mutator phenotype in the presence of the wild-type allele is consistent with the participation of both the wild-type and the mutant polymerases in DNA replication, in contrast to mutated MMR tumors, where loss of both alleles is required to produce a mutator effect.It has been proposed that differential expression levels of the wild-type and mutant POLE alleles in the course of cancer progression may allow transient stages of hypermutation that promote tumor growth together with a threshold limiting excessive mutation load to maintain fitness.
Finally, we thank the reviewer who commented on a new original concept in Figure 1 and the consequences for new treatment options for specific cancers.Thank you for submitting your revised manuscript entitled "Exploring the basis of heterogeneity of cancer aggressiveness among the mutated POLE variants".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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You can contact the journal office with any questions, contact@life-science-alliance.orgAgain, congratulations on a very nice paper.I hope you found the review process to be constructive and are pleased with how the manuscript was handled editorially.We look forward to future exciting submissions from your lab.Sincerely, Eric Sawey, PhD Executive Editor Life Science Alliance http://www.lsajournal.org to the reviewers for their constructive comments and suggestions on our review.Based on their comments, we changed critical parts throughout the manuscript.We have particularly included original citations and adequate references that were missing in the initial manuscript.We apologize for this weakness in the previous version of the paper and we agree that for a review, the citations are a critical component.The new parts of the text are highlighted in blue.Please see below our detailed point by point response to the specific comments of each reviewer:Reviewer #1:Paragraph POLE mutations and cancer 1. Major Criticism: http://www.lsajournal.org---------------------------------------------------------------------------submitting your Review entitled "Exploring the basis of heterogeneity of cancer aggressiveness among the mutated POLE variants".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.